ABSTRACTCellular uptake of the human immunodeficiency virus TAT protein transduction domain (PTD), or cell-penetrating peptide, has previously been surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans that are expressed ubiquitously on the cell surface. These acidic polysaccharides form a large pool of negative charge on the cell surface that TAT PTD binds avidly. Additionally, sulfated glycans have been proposed to aid in the interaction of TAT PTD and other arginine-rich PTDs with the cell membrane, perhaps aiding their translocation across the membrane. Surprisingly, however, TAT PTD-mediated induction of macropinocytosis and cellular transduction occurs in the absence of heparan sulfate and sialic acid. Using labeled TAT PTD peptides and fusion proteins, in addition to TAT PTD-Cre recombination-based phenotypic assays, we show that transduction occurs efficiently in mutant Chinese hamster ovary cell lines deficient in glycosaminoglycans and sialic acids. Similar results were obtained in cells where glycans were enzymatically removed. In contrast, enzymatic removal of proteins from the cell surface completely ablated TAT PTD-mediated transduction. Our findings support the hypothesis that acidic glycans form a pool of charge that TAT PTD binds on the cell surface, but this binding is independent of the PTD-mediated transduction mechanism and the induction of macropinocytotic uptake by TAT PTD.

Figure 4: Macropinocytotic inhibitors and TAT PTD-Cre transduction in glycan-deficient cells. A, CHO-K1, pgsA, and Lec2 cells with a stably integrated LSL-GFP construct treated with the indicated concentrations of amiloride, an inhibitor of macropinocytosis, for 1 h. Cells were then treated with TAT PTD-Cre protein in the presence of amiloride for 1 h, followed by trypsinization and replating. GFP expression was assayed by flow cytometry at 24 h after TAT PTD-Cre addition. Inset, cell viability as assayed by flow cytometry immediately following TAT PTD-Cre treatment. B, treatment with wortmannin, a kinase inhibitor, as described for A. C, treatment with cytochalasin D, an inhibitor of F-actin and macropinocytosis, as described for A. D, depletion of cell-surface proteins by trypsin. CHO-K1 LSL cells were treated with trypsin or cell dissociation solution (CDS) for 30 min, followed by washes with PBS and trypsin inhibitor and treatment with TAT PTD-Cre for 1 h. Cells were then washed, trypsinized, and replated. GFP expression was assayed by FACS at 24 h following TAT PTD-Cre treatment. WT, wild-type.

Figure 4: Macropinocytotic inhibitors and TAT PTD-Cre transduction in glycan-deficient cells. A, CHO-K1, pgsA, and Lec2 cells with a stably integrated LSL-GFP construct treated with the indicated concentrations of amiloride, an inhibitor of macropinocytosis, for 1 h. Cells were then treated with TAT PTD-Cre protein in the presence of amiloride for 1 h, followed by trypsinization and replating. GFP expression was assayed by flow cytometry at 24 h after TAT PTD-Cre addition. Inset, cell viability as assayed by flow cytometry immediately following TAT PTD-Cre treatment. B, treatment with wortmannin, a kinase inhibitor, as described for A. C, treatment with cytochalasin D, an inhibitor of F-actin and macropinocytosis, as described for A. D, depletion of cell-surface proteins by trypsin. CHO-K1 LSL cells were treated with trypsin or cell dissociation solution (CDS) for 30 min, followed by washes with PBS and trypsin inhibitor and treatment with TAT PTD-Cre for 1 h. Cells were then washed, trypsinized, and replated. GFP expression was assayed by FACS at 24 h following TAT PTD-Cre treatment. WT, wild-type.

ABSTRACTCellular uptake of the human immunodeficiency virus TAT protein transduction domain (PTD), or cell-penetrating peptide, has previously been surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans that are expressed ubiquitously on the cell surface. These acidic polysaccharides form a large pool of negative charge on the cell surface that TAT PTD binds avidly. Additionally, sulfated glycans have been proposed to aid in the interaction of TAT PTD and other arginine-rich PTDs with the cell membrane, perhaps aiding their translocation across the membrane. Surprisingly, however, TAT PTD-mediated induction of macropinocytosis and cellular transduction occurs in the absence of heparan sulfate and sialic acid. Using labeled TAT PTD peptides and fusion proteins, in addition to TAT PTD-Cre recombination-based phenotypic assays, we show that transduction occurs efficiently in mutant Chinese hamster ovary cell lines deficient in glycosaminoglycans and sialic acids. Similar results were obtained in cells where glycans were enzymatically removed. In contrast, enzymatic removal of proteins from the cell surface completely ablated TAT PTD-mediated transduction. Our findings support the hypothesis that acidic glycans form a pool of charge that TAT PTD binds on the cell surface, but this binding is independent of the PTD-mediated transduction mechanism and the induction of macropinocytotic uptake by TAT PTD.